The present invention relates to the production of paucilamellar lipid vesicles having charge-localized, single chain nonphospholipid zwitterionic or anionic surfactants as the primary structural material of their lipid bilayers. More particularly, the present invention relates to a method of producing these paucilamellar lipid vesicles having a large aqueous or organic liquid filled amorphous central cavity, as well as the vesicles themselves.
Lipid vesicles are substantially spherical structures made of materials having a high lipid content, e.g., surfactants or phospholipids. The lipids of these spherical vesicles are organized in the form of lipid bilayers. The lipid bilayers encapsulate an aqueous volume which is either interspersed between multiple onion-like shells of lipid bilayers, forming a classic multilamellar lipid contained within an amorphous central cavity. Common lipid vesicles having an amorphous central cavity filled with aqueous medium are the unilamellar lipid vesicles. Large unilamellar vesicles ("LUV"'s) generally have a diameter greater than about 1 .mu. while small unilamellar lipid vesicles ("SUV"'s) generally have a diameter of less than 0.2 .mu..
Paucilamellar lipid vesicles ("PLV"'s) are a hybrid having features of both MLV's and LUV's. PLV's are characterized by having 2-10 peripheral bilayers surrounding a large, unstructured central cavity.
The potential utility of liposomes is widely recognized. Their ability to encapsulate aqueous volumes and/or lipophilic material makes them attractive devices for transporting a whole spectrum of molecules, including macromolecules and drugs, vaccines, and other therapeutic compositions. In addition, it is possible to encapsulate supramolecular structures such as viruses using classes of liposomes. Some types of vesicles have shown an ability to act as adjuvants or as carriers or storage devices for oil-based materials.
Each type of lipid vesicle appears to have certain uses for which it is best adapted. For example, the multiple onion-like lipid bilayers of classic MLV's provide this lipid vesicle with increased durability and protection from enzymatic degradation. The multiple shells greatly diminish the volume available for aqueous solutions to be encapsulated within the bilayers of the MLV. MLV's have heretofore been deemed most advantageous for carrying lipophilic materials which can be incorporated in their bilayers. However, there is a maximum amount of lipophilic material that can be incorporated into MLV bilayers, beyond which the bilayers become unstable and these vesicles break down. In contrast, the single shell of LUV's allow the encapsulation of a larger volume of aqueous material but because of their single lipid bilayer structure, LUV's are not as physically durable as MLV's. SUV's have neither the lipid or aqueous volumes of MLV's or LUV's, but because of their small size have easiest access to cells and tissues.
PLV's appear to have advantages as transport vehicles for many uses as compared with the other types of lipid vesicles. In particular, because of their large unstructured central cavity, PLV's are easily adapted for transport of large quantities of aqueous-based materials. However, their multiple lipid bilayers provide PLV's with the ability to carry lipophilic material in their bilayers as well as with additional physical strength and resistance to degradation as compared with the single lipid bilayer of the LUV. In addition, as illustrated in the present application and U.S. patent application Ser. No. 157,571, now U.S. Pat. No. 4,911,928, the disclosure of which is incorporated herein by reference, the central cavity of the PLV's can be filled wholly or in part with an apolar oil or wax and then can be used as a vehicle for the transport or storage of hydrophobic materials. Thus, the amount of hydrophobic material which can be transported by PLV's with an apolar core is much greater than can be transported by classic MLV's.
Early lipid vesicle or liposome studies used phospholipids as the lipid source for bilayers, primarily because phospholipids are the principal structural components of natural membranes. However, there are a number of problems associated with using phospholipids as artificial membranes. First, isolated phospholipids are subject to degradation by a large variety of enzymes. Second, the most easily available phospholipids are those from natural sources, e.g., egg yolk lecithin, which contain polyunsaturated acyl chains that are subject to autocatalyzed peroxidation. When peroxidation occurs, the lipid structure breaks down, causing premature release of encapsulated materials and the formation of toxic peroxidation byproducts. This problem can be avoided by hydrogenation but hydrogenation is an expensive process, thereby raising the cost of the starting materials. Cost is a third problem associated with the use of phospholipids on a large scale. The high cost of a kilogram of egg yolk lecithin pure enough for pharmacological liposome production places a severe limitation on the use of phospholipids as a source material.
It is now known that commercially available surfactants may be used to form the lipid bilayer in a variety of lipid vesicles. (See, e.g., U.S. Pat. No. 4,217,344, U.S. Pat. No. 4,855,090, and U.S. patent application Ser. No. 157,571 now U.S. Pat. No. 4,911,928). Both surfactants and phospholipids are amphiphiles, having at least one lipophilic acyl or alkyl group attached to a hydrophilic head group. The head groups are attached to one or more lipophilic chains by ester, ether or amide linkages. Commercially available surfactants include the BRIJ family of polyoxyethylene fatty acid ethers, the SPAN sorbitan alkyl esters, and the TWEEN polyoxyethylene sorbitan fatty acid esters, all available from ICI Americas, Inc., of Wilmington, Delaware. Unlike phospholipids, these surfactants are generally nonionic, and addition of a charge-producing amphiphile is usually required to prevent floculation and to increase the degree of encapsulation of water-soluble substances. Addition of a charge-producing amphiphile is not required if the primary wall lipid is anionic--as in the case of sarcosinamides.
In addition, the presence of a sterol or sterol-like molecule in the lipophilic phase used to create the lipid bilayer has often been found to be important for increasing the stability of the bilayer and hence the vesicle.
In 1982, Murakami et al. disclosed the preparation of small (0.05-0.2 .mu.), single- or multiple-walled vesicles capable of encapsulating aqueous volumes, using cationic and zwitterionic double chain amphiphiles synthesized to mimic the structure of naturally occurring phospholipids. The aqueous-carrying capacity of these vesicles is unknown as is their structure and stability. Murakami does not mention the possibility of oil encapsulation or single chain varieties of these ionic lipids.
The use of anionic or zwitterionic surfactants as cleaning or conditioning agents in cleansers such as shampoos is well documented in the art (see e.g., U.S. Pat. No. 4,075,131 and U.S. Pat. No. 4,832,872). However, in their present formulation in cleanser compositions, these surfactants are not present in vesicle form.
Recently an improved method for creating large aqueous volume MLV's and PLV's using commercially available, synthethic, nonionic surfactants has been discovered. U.S. Pat. No. 4,855,090, and U.S. patent application Ser. No. 157,571 now U.S. Pat. No. 4,911,928, disclose this new method which has the advantage of being faster and more cost-efficient than previous methods. This improved method of creating PLV's and large aqueous volume MLV's, which is applicable to only certain surfactants, forms vesicles in less than a second rather than the minutes or hours of classical techniques. Moreover, the improved method allows vesicles to be formed without the use of solvents and without the formation of a separable lamellar phase. These techniques, and the devices to utilize them, have only been described in the aforementioned patents, as well as the related applications. In contrast, the classic methods for producing multilamellar lipid vesicles are well-documented in the art. See for example Gregoriadis, G., ed. Liposome Technology (CRC, Boca Raton, FL), Vols. 1-3 (1984), and Dousset and Douste-Blzay (in Les Liposomes, Puisieux and Delattre, ed., Techniques et Documentation Lavoiser, Paris, pp. 41-73 (1985).
No matter how the MLV's or PLV's are formed, once made it is necessary to determine the effectiveness of the process. Two measurements commonly used to determine the effectiveness of encapsulation of materials in lipid vesicles are the encapsulated mass and captured volume. The encapsulated mass is the mass of the substance encapsulated per unit mass of the lipid and is often given as a percentage. The captured volume is defined as the amount of the aqueous phase trapped inside the vesicle divided by the amount of lipid in the vesicle structure, normally given in ml liquid/g lipid.
The methods and materials disclosed herein for producing paucilamellar lipid vesicles formed of single chain charge-localized nonphospholipid zwitterionic or anionic surfactants all yield stable vesicles capable of encapsulating aqueous or oil volumes.
Accordingly, an object of the invention is to provide stable paucilamellar lipid vesicles from charge-localized non-phospholipid single chain surfactants.
Another object of the invention is to provide a method for producing such paucilamellar lipid vesicles which is rapid and uses relatively inexpensive materials.
A further object of the invention is to provide a vehicle for the transport of aqueous or oil-soluble materials formed essentially of charge-localized nonphospholipid single chain zwitterionic or anionic surfactants.
These and other objects and features of the invention will be apparent from the detailed description and the claims.